Hepatitis B virus core protein can self-assemble into icosahedral symmetrical viral-like particles (VLPs) in vitro, and display exogenous sequences repeatedly and densely on the surface. VLPs also have strong immunogenicity and biological activity. When the nanoparticles enter the body, they quickly induce specific humoral and cellular immune responses to exogenous antigens. In this study, we designed an HBc-VLPs that can be coupled with antigens at specific sites, and developed a set of efficient methods to prepare HBc-VLPs. Through site-specific mutation technology, the 80th amino acid of peptide was changed from Ala to Cys, a specific cross-linking site was inserted into the main immunodominant region of HBc-VLPs, and the prokaryotic expression vector pET28a(+)-hbc was constructed. After expression and purification, high purity HBc(A80C) monomer protein was assembled into HBc-VLPs nanoparticles in Phosphate Buffer. The results of particle size analysis show that the average particle size of nanoparticles was 29.8 nm. Transmission electron microscopy (TEM) showed that HBc-VLPs formed spherical particles with a particle size of about 30 nm, and its morphology was similar to that of natural HBV particles. The influenza virus antigen M2e peptide as model antigen was connected to Cys residue of HBc-VLPs by Sulfo-SMCC, an amino sulfhydryl bifunctional cross-linking agent, and M2e-HBc-VLPs model vaccine was prepared. The integrity of HBc-VLPs structure and the correct cross-linking of M2e were verified by cell fluorescence tracing. Animal immune experiments showed that the vaccine can effectively stimulate the production of antigen-specific IgG antibody in mice, which verified the effectiveness of the vaccine carrier HBc-VLPs. This study lays a foundation for the research of HBc-VLPs as vaccine vector, and help to promote the development of HBc-VLPs vaccine and the application of HBc-VLPs in other fields.
Animals ; Hepatitis B Core Antigens ; genetics ; immunology ; Immunity, Cellular ; immunology ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Vaccines, Virus-Like Particle ; genetics ; immunology

Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β (IFN-β). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector-IFN-β transcripts under RIG-I' ligand, 5'-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection.
Animals ; Cell Line, Tumor ; Cloning, Molecular ; DEAD Box Protein 58 ; antagonists & inhibitors ; genetics ; immunology ; Fibroblasts ; immunology ; pathology ; Gene Expression ; Hepatitis B ; genetics ; immunology ; pathology ; veterinary ; Hepatitis B Virus, Woodchuck ; Immunity, Innate ; Interferon-beta ; genetics ; immunology ; Isoelectric Point ; Kidney ; immunology ; pathology ; virology ; Liver ; immunology ; pathology ; virology ; Marmota ; genetics ; immunology ; virology ; Open Reading Frames ; Protein Domains ; RNA, Double-Stranded ; RNA, Small Interfering ; genetics ; metabolism ; Rodent Diseases ; genetics ; immunology ; pathology ; virology

Bone marrow stromal antigen 2 (BST-2) is a kind of host restriction factor. Since it was discovered to be responsible for the defect in virion release of HIV-1 mutants lacking the accessory gene vpu in 2008, it was thought to mainly restrict the viruses by directly tethering viral particles at the plasma membrane. Recent reports suggest that BST-2 also can inhibit the the release of HBV particles, which are budding in the intracellular vesicles, expanding the antiviral spectrum of BST-2. Futhermore, the machanism that BST-2 used to restrict HBV release in multivesicular bodies (MVBs) is similar to that used to restrict HIV at the plasma membrane. However, HBV have evolved strategies to antagonize the antiviral action of BST-2. There are two different opinions about the antagonist. One is HBV inactivated BST-2 by HBx requiring a hepatocyte-specific environment. Another thought envelope protein HBs counteract the antiviral action of BST-2. In this review, we focus on the current advances in the anti-HBV activity of BST-2.
Animals ; Antigens, CD ; genetics ; immunology ; GPI-Linked Proteins ; genetics ; immunology ; Hepatitis B ; genetics ; immunology ; virology ; Hepatitis B virus ; genetics ; physiology ; Host-Pathogen Interactions ; Humans ; Virus Release

PURPOSE: Acute hepatitis A (AHA) and acute hepatitis B (AHB) are caused by an acute infection of the hepatitis A virus and the hepatitis B virus, respectively. In both AHA and AHB, liver injury is known to be mediated by immune cells and cytokines. In this study, we measured serum levels of various cytokines and T-cell cytotoxic proteins in patients with AHA or AHB to identify liver injury-associated cytokines. MATERIALS AND METHODS: Forty-six patients with AHA, 16 patients with AHB, and 14 healthy adults were enrolled in the study. Serum levels of 17 cytokines and T-cell cytotoxic proteins were measured by enzyme-linked immunosorbent assays or cytometric bead arrays and analyzed for correlation with serum alanine aminotransferase (ALT) levels. RESULTS: Interleukin (IL)-18, IL-8, CXCL9, and CXCL10 were significantly elevated in both AHA and AHB. IL-6, IL-22, granzyme B, and soluble Fas ligand (sFasL) were elevated in AHA but not in AHB. In both AHA and AHB, the serum level of CXCL10 significantly correlated with the peak ALT level. Additionally, the serum level of granzyme B in AHA and the serum level of sFasL in AHB correlated with the peak ALT level. CONCLUSION: We identified cytokines and T-cell cytotoxic proteins associated with liver injury in AHA and AHB. These findings deepen the existing understanding of immunological mechanisms responsible for liver injury in acute viral hepatitis.
Acute Disease ; Adult ; Alanine Transaminase/blood ; Biomarkers/blood ; Cytokines/*blood ; Enzyme-Linked Immunosorbent Assay ; Fas Ligand Protein/blood ; Female ; Hepatitis A/blood/virology ; Hepatitis A virus/*genetics/immunology ; Hepatitis B/blood/virology ; Hepatitis B virus/*genetics/immunology ; Humans ; Interleukin-6/blood ; Interleukin-8/blood ; Interleukins/blood ; Liver Failure/immunology/metabolism/*pathology ; Male ; Middle Aged ; T-Lymphocytes, Cytotoxic/immunology/*metabolism

BACKGROUND/AIMS: The efficacy of tenofovir disoproxil fumarate (TDF) for the treatment of chronic hepatitis B (CHB) patients following prior treatment failure with multiple nucleos(t)ide analogues (NAs) is not well defined, especially in Asian populations. In this study we investigated the efficacy and safety of TDF rescue therapy in CHB patients after multiple NA treatment failure. METHODS: The study retrospectively analyzed 52 CHB patients who experienced failure with two or more NAs and who were switched to regimens containing TDF. The efficacy and safety assessments included hepatitis B virus (HBV) DNA undetectability, hepatitis B envelop antigen (HBeAg) seroclearance, alanine transaminase (ALT) normalization and changes in serum creatinine and phosphorus levels. RESULTS: The mean HBV DNA level at baseline was 5.4 +/- 1.76 log10 IU/mL. At a median duration of 34.5 months of TDF treatment, the cumulative probabilities of achieving complete virological response (CVR) were 25.0%, 51.8%, 74.2%, and 96.7% at 6, 12, 24, and 48 months, respectively. HBeAg seroclearance occurred in seven of 48 patients (14.6%). ALT levels were normalized in 27 of 31 patients (87.1%) with elevated ALT at baseline. Lower levels of HBV DNA at baseline were significantly associated with increased CVR rates (p < 0.001). However, CVR rates did not differ between TDF monotherapy or combination therapy with other NAs, and were not affected by mutations associated with resistance to NAs. No significant adverse events were observed. CONCLUSIONS: TDF is an efficient and safe rescue therapy for CHB patients after treatment failure with multiple NAs.
Adenine/adverse effects/*analogs & derivatives/therapeutic use ; Adult ; Aged ; Alanine Transaminase/blood ; Antiviral Agents/adverse effects/*therapeutic use ; Biological Markers/blood ; Creatinine/blood ; DNA, Viral/blood ; Drug Resistance, Viral/genetics ; Drug Substitution ; Female ; Genotype ; Hepatitis B e Antigens/blood ; Hepatitis B virus/*drug effects/genetics/immunology/pathogenicity ; Hepatitis B, Chronic/blood/diagnosis/*drug therapy ; Humans ; Kaplan-Meier Estimate ; Male ; Middle Aged ; Mutation ; Phosphorous Acids/adverse effects/*therapeutic use ; Phosphorus/blood ; Retrospective Studies ; Time Factors ; Treatment Failure ; Viral Load ; Young Adult

PURPOSE: The role of IL28B gene variants and expression in hepatitis B virus (HBV) infections are not well understood. Here, we evaluated whether IL28B gene expression and rs12979860 variations are associated with HBV outcomes. MATERIALS AND METHODS: IL28B genetic variations (rs12979860) were genotyped by pyrosequencing of DNA samples from 137 individuals with chronic HBV infection [50 inactive carriers (IC), 34 chronic hepatitis B (CHB), 27 cirrhosis, 26 hepatocellular carcinoma (HCC)], and 19 healthy controls. IL28A/B mRNA expression in peripheral blood mononuclear cells was determined by qRT-PCR, and serum IL28B protein was measured by ELISA. RESULTS: Patients with IL28B C/C genotype had greater IL28A/B mRNA expression and higher IL28B protein levels than C/T patients. Within the various disease stages, compared to IC and healthy controls, IL28B expression was reduced in the CHB, cirrhosis, and HCC cohorts (CHB vs. IC, p=0.02; cirrhosis vs. IC, p=0.01; HCC vs. IC, p=0.001; CHB vs. controls, p<0.01; cirrhosis vs. controls, p<0.01; HCC vs. controls, p<0.01). When stratified with respect to serum HBV markers in the IC and CHB cohorts, IL28B mRNA and protein levels were higher in HBeAg-positive than negative individuals (p=0.01). Logistic regression analysis revealed that factors associated with high IL28B protein levels were C/C versus C/T genotype [p=0.016, odds ratio (OR)=0.25, 95% confidence interval (CI)=0.08-0.78], high alanine aminotransferase values (p<0.001, OR=8.02, 95% CI=2.64-24.4), and the IC stage of HBV infection (p<0.001). CONCLUSION: Our data suggest that IL28B genetic variations may play an important role in long-term development of disease in chronic HBV infections.
Adult ; Aged ; Alanine Transaminase/blood ; Asian Continental Ancestry Group/*genetics ; Biological Markers/blood ; Carcinoma, Hepatocellular/genetics ; Case-Control Studies ; China ; DNA, Viral/blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Genotype ; Hepatitis B virus/genetics ; Hepatitis B, Chronic/ethnology/*genetics/immunology/*virology ; Humans ; Interleukins/blood/*genetics/metabolism ; Leukocytes, Mononuclear ; Liver Cirrhosis/blood ; Liver Neoplasms/genetics ; Male ; Middle Aged ; RNA, Messenger/*genetics ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUND/AIMS: Quantification of hepatitis B surface antigen (HBsAg) is an emerging serologic test and may be useful for identifying treatment strategies for chronic hepatitis B (CHB). This study aimed to evaluate HBsAg titers during the natural course of CHB and identify correlations between HBsAg titers and hepatitis B virus (HBV) DNA concentrations across different CHB phases measured using an immunoradiometric assay (IRMA). METHODS: CHB phases were defined on the basis of HBV DNA concentrations, the presence of hepatitis B e antigen/antibody (HBeAg/Ab) and serum alanine aminotransferase levels. Serum HBsAg titers and paired HBV DNA concentrations in the different phases of CHB were compared using 627 serum samples. RESULTS: Mean HBsAg titers were significantly higher in the immunotolerant (IT) phase and immunoreactive (IR) HBeAg-positive phase than in the low-replicative (LR) and HBeAg-negative CHB (ENH) states. The correlation between HBsAg titers and HBV DNA concentrations was modest in the IT (n=36, r=0.804, p<0.001) and IR (n=48, r=0.773, p<0.001) phases, and it was poor in the LR state (n=116, r=0.289, p=0.002); however, no significant correlation was observed in the ENH state (n=67, r=0.146, p=0.237) or in the oral nucleos(t)ide analogue-treated group (n=267). CONCLUSIONS: HBsAg quantification using IRMA might be useful for discriminating different CHB phases and different stages of chronic liver disease.
Adult ; Alanine Transaminase/blood ; Biomarkers/blood ; DNA, Viral/*blood ; Disease Progression ; Female ; Hepatitis B Surface Antigens/*blood ; Hepatitis B e Antigens/blood ; Hepatitis B virus/*genetics/immunology ; Hepatitis B, Chronic/*immunology ; Humans ; *Immunoradiometric Assay ; Male ; Middle Aged ; Seoul ; Viral Load ; Virus Replication

<b>BACKGROUNDb>Some hepatitis B extracellular antigen (HBeAg)-positive chronic hepatitis B (CHB) patients in their immune active phase can clear the virus spontaneously and enter into an inactive hepatitis B virus (HBV) carrier state, indicating a benign prognosis. In this study, the association between cytokine-inducible SRC homology 2 domain protein (CISH) gene polymorphisms at -292 (rs414171) and the spontaneous clearance of HBV in HBeAg-positive CHB patients in immune the active phase was investigated.

<b>METHODSb>Seventy HBeAg-positive CHB patients in the immune active phase were followed up for 76 weeks without antiviral therapy. The alanine transaminase, aspartate transaminase, HBV DNA, HBeAg and hepatitis B extracellular antibody levels were tested regularly. At week 76, 27 patients were classified into group A (HBV DNA level below 2 104 IU/ml and the value of HBeAg declined below 10% of the baseline at week 76), and 43 patients were classified into group B (HBV DNA level higher than 2×10(4) IU/ml or the value of HBeAg did not decline substantially at week 76). CISH (rs414171) polymorphisms were also tested using the iPLEX system.

<b>RESULTSb>The HBV DNA levels at week 12 were significantly greater in group B compared with group A (group A: (6.87±1.40) log10IU/ml; group B: (7.61±1.38) log10IU/ml, P = 0.034) and the HBeAg values were greater in group B at week 28 compared with group A (P = 0.001). The differences in HBV DNA and HBeAg values increased between the groups over time. Sixteen patients in group A and 11 in group B were genotype AA. Those with genotype AT or TT included 11 in group A and 31 in group B (AA vs. AT and TT, odds ratio 4.10 (95% confidence interval: 1.462-11.491), P = 0.006).

<b>CONCLUSIONb>CISH gene polymorphisms at -292 (rs414171) are associated with HBV clearance in HBeAg-positive CHB patients in the immune active phase, and AA is a favorable genotype for this effect.

Adolescent ; Adult ; Aged ; Female ; Genotype ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; immunology ; Hepatitis B, Chronic ; immunology ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; genetics ; Suppressor of Cytokine Signaling Proteins ; genetics ; Young Adult

<b>OBJECTIVEb>To explore the effect of compound qizhu granule (CQG) on cellular immunity of chronic hepatitis B (CHB) patients.

<b>METHODSb>Totally 103 CHB patients treated with lamivudin (LAM) for 6 months, who had partial virological response (HBeAg positive) were randomly assigned to two groups, 50 in the treatment group and 53 in the control group. All patients took LAM 100 mg (once a day) plus ADV 10 mg (once a day). Patients in the treatment group additionally took CQG, one dose per day. After one-year treatment hepatitis B virus (HBV) DNA negative rates, HBeAg seroconversion, levels of HBV specific cytotoxic T lymphocyte (CTL), non-specific CTL and natural killing (NK) cells were compared between the two groups.

<b>RESULTSb>After 1-year treatment, HBV DNA negative rate of the treatment group was 88: 0% in 44 cases, slightly higher than that of the control group (41 cases, 77.4%), but with no statistical difference (P >0.05). HBeAg seroconversion of the treatment group was 32.0% in 16 cases, higher than that of the control group (8 cases, 15.1%), with statistical difference (P <0.05). Levels of HBV specific CTL (0.79%±0. 07%), non-specific CTL (19.4%±1.8%) and NK cells (14. 1%± 1.5%) of the treatment group were higher than those of the control group (0.58% ± 0.08%, 17.5% ± 1.7%, and 11.1%±1.5%, respectively; allP <0.01).

<b>CONCLUSIONb>Treating CHB patients with partial virological response by ADV plus CQG could improve specific and non-specific cellular immunity, thereby elevating HBeAg seroconversion rate.

Drugs, Chinese Herbal ; therapeutic use ; Hepatitis B e Antigens ; immunology ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; drug therapy ; immunology ; Humans ; Immunity, Cellular ; immunology ; T-Lymphocytes, Cytotoxic ; drug effects

<b>OBJECTIVEb>To investigate the effect of protein transduction domain-hepatitis B virus core antigen (CTP-HBcAg18-27)-Tapasin fusion protein-induced specific cytotoxic T lymphocyte (CTL) response on hepatitis B virus (HBV) replication in HBV transgenic mice.

<b>METHODSb>Twenty HBV-transgenic mice were randomly divided into two groups for a 3-week course of once weekly subcutaneous immunizations with either CTP-HBcAg18-27-Tapasin fusion protein or CTP-HBcAg18-27. Mice administered isotonic saline served as blank controls. Expressions of cytokines in splenocytes were analyzed by flow cytometry. Serum levels of hepatitis B surface antigen (HBsAg) and HBV DNA were determined by microparticle enzyme immunoassay and real-time fluorescent PCR assay, respectively. Expression of HBsAg in hepatic tissues was detected by immunohistochemistry.

<b>RESULTSb>Immunization with 100 mug of CTP-HBcAg18-27-Tapasin fusion protein led to a significant increase in proportions of CTLs in spleen (2.70%+/-0.20% vs. 50 mug of CTP-HBcAg18-27-Tapasin: 1.66%+/-0.53%, 50 mug of CTP-HBcAg18-27: 1.26%+/-0.56%, and blank controls: 0.75%+/-0.71%; F = 741.45, P = 0.000) and up-regulation of inflammatory cells in hepatic tissue. In addition, both immunizations of CTP-HBcAg18-27-Tapasin led to significant decreases in serum HBsAg and HBV DNA levels compared to those in the CTP-HBcAg18-27 group.

<b>CONCLUSIONb>HBV-related modification of the expression of the molecular chaperone Tapasin may affect its interaction with intracellular antigen peptides, thereby leading to increases the number of specific CTLs in the spleen, decreases in serum HBsAg and HBV DNA levels, and down-regulation of HBsAg expression in hepatic tissue. These results obtained in HBV-transgenic mice suggest that the CTP-HBcAg18-27-Tapasin fusion protein has anti-HBV activity.

Animals ; DNA, Viral ; blood ; Female ; Hepatitis B ; immunology ; Hepatitis B Core Antigens ; genetics ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; physiology ; Male ; Membrane Transport Proteins ; genetics ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Virus Replication